Small ruminant macrophage polarization may play a pivotal role on lentiviral infection

Small ruminant lentiviruses (SRLV) infect the monocyte/macrophage lineage inducing a long-lasting infection affecting body condition, production and welfare of sheep and goats all over the world. Macrophages play a pivotal role on the host's innate and adaptative immune responses against parasites by becoming differentially activated. Macrophage heterogeneity can tentatively be classified into classically differentiated macrophages (M1) through stimulation with IFN-gamma displaying an inflammatory profile, or can be alternatively differentiated by stimulation with IL-4/IL-13 into M2 macrophages with homeostatic functions. Since infection by SRLV can modulate macrophage functions we explored here whether ovine and caprine macrophages can be segregated into M1 and M2 populations and whether this differential polarization represents differential susceptibility to SRLV infection. We found that like in human and mouse systems, ovine and caprine macrophages can be differentiated with particular stimuli into M1/M2 subpopulations displaying specific markers. In addition, small ruminant macrophages are plastic since M1 differentiated macrophages can express M2 markers when the stimulus changes from IFN-gamma to IL-4. SRLV replication was restricted in M1 macrophages and increased in M2 differentiated macrophages respectively according to viral production. Identification of the infection pathways in macrophage populations may provide new targets for eliciting appropriate immune responses against SRLV infection.This work was funded by grants from CICYT (no. AGL2010-22341-C04-01), and the Government of Navarra (no. IIQ14064.RI1). We acknowledge the Public University of Navarra and CSIC for fellowships and the JAE-contract (HC and RR)

Mannose receptor may be involved in small ruminant lentivirus pathogenesis

Thirty-one sheep naturally infected with small ruminant lentiviruses (SRLV) of known genotype (A or B), and clinically affected with neurological disease, pneumonia or arthritis were used to analyse mannose receptor (MR) expression (transcript levels) and proviral load in virus target tissues (lung, mammary gland, CNS and carpal joints). Control sheep were SRLV-seropositive asymptomatic (n = 3), seronegative (n = 3) or with chronic listeriosis, pseudotuberculosis or parasitic cysts (n = 1 in each case). MR expression and proviral load increased with the severity of lesions in most analyzed organs of the SRLV infected sheep and was detected in the affected tissue involved in the corresponding clinical disease (CNS, lung and carpal joint in neurological disease, pneumonia and arthritis animal groups, respectively). The increased MR expression appeared to be SRLV specific and may have a role in lentiviral pathogenesis.Funded by grants from CICYT AGL2010-22341-C04-01 and Gobierno de Navarra IIQ14064.RI1. We acknowledge the Public University of Navarra and CSIC for fellowships and the JAE-contract (HC and RR)

Post-entry blockade of small ruminant lentiviruses by wild ruminants

Small ruminant lentivirus (SRLV) infection causes losses in the small ruminant industry due to reduced animal production and increased replacement rates. Infection of wild ruminants in close contact with infected domestic animals has been proposed to play a role in SRLV epidemiology, but studies are limited and mostly involve hybrids between wild and domestic animals. In this study, SRLV seropositive red deer, roe deer and mouflon were detected through modified ELISA tests, but virus was not successfully amplified using a set of different PCRs. Apparent restriction of SRLV infection in cervids was not related to the presence of neutralizing antibodies. In vitro cultured skin fibroblastic cells from red deer and fallow deer were permissive to the SRLV entry and integration, but produced low quantities of virus. SRLV got rapidly adapted in vitro to blood-derived macrophages and skin fibroblastic cells from red deer but not from fallow deer. Thus, although direct detection of virus was not successfully achieved in vivo, these findings show the potential susceptibility of wild ruminants to SRLV infection in the case of red deer and, on the other hand, an in vivo SRLV restriction in fallow deer. Altogether these results may highlight the importance of surveilling and controlling SRLV infection in domestic as well as in wild ruminants sharing pasture areas, and may provide new natural tools to control SRLV spread in sheep and goats.Funded by CICYT (AGL2010-22341-C04-01 and AGL2013-49137-C3-1-R) and Navarra’s Government (IIQ010449.RI1 and IIQ14064.RI1). L. Sanjosé was a FPI fellow of the Spanish MINECO and R. Reina had contracts from the Public University of Navarra and CSIC. The auhors acknowledge support in the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)

Base genética del tropismo de lentivirus de pequeños rumiantes y estudio de la resistencia innata por APOBEC3

Esta tesis doctoral ha sido realizada dentro de los siguientes proyectos de investigación: AGL2003‐08977‐C03‐01, AGL2006‐13410‐C06‐01/GAN, AGL2007‐66874‐C04‐01/GAN, AGL2010‐22341‐C04‐01, y han sido financiados por la Comisión Interministerial de Ciencia y Tecnología (CICYT).Los lentivirus de pequeños rumiantes (SRLV), que incluyen al virus Visna/Maedi (VMV) y al de la artritis encefalitis caprina (CAEV), infectan ovejas y cabras distribuidas por todo el mundo causando un cuadro multisistémico que afecta articulaciones, pulmones, glándula mamaria y sistema nervioso central. Las pérdidas derivadas de la infección van desde el aumento en la tasa de reposición, a un descenso en las producciones animales o en el valor comercial del rebaño. Aunque la enfermedad causada por los SRLV es de carácter lento y generalmente afecta a pulmones y glándula mamaria en nuestro país, se han descrito brotes epidemiológicos causantes de artritis y encefalitis en ovinos que afectan un gran número de animales, causando pérdidas directas. En la actualidad existen 5 genotipos descritos (A‐E) que presentan una alta variabilidad genética y biológica. Los genotipos A y B a los que pertenecen las estirpes clásicas de VMV y CAEV respectivamente, están distribuidos mundialmente, mientras que los genotipos C y E están restringidos a zonas geográficas concretas. En ausencia de tratamientos o vacunas totalmente protectoras, las medidas de control se basan en la detección temprana de animales infectados y su posterior eliminación del rebaño. Tras la infección, los animales infectados desarrollan una respuesta de anticuerpos que, si bien no es capaz de eliminar el virus, es indicadora de la infección. Así, empleando métodos de detección serológica podemos diagnosticar la infección indirectamente. Los métodos más eficaces hasta el momento son los ELISAs basados en proteínas recombinantes y péptidos sintéticos. Sin embargo, los métodos disponibles en el comercio están diseñados teniendo en cuenta una única estirpe viral, que sumado a la alta variabilidad antigénica de los SRLV, hace que las medidas disponibles fallen a la hora de controlar todo el espectro antigénico presente. La organización genómica de los lentivirus está bastante conservada entre los miembros del género. Así, a los genes estructurales gag, pol y env encargados de codificar proteínas de la cápside, proteínas para la replicación del material genético e integración y las proteínas de la envoltura, respectivamente, hay que sumarles los accesorios vpr, rev y vif en el caso de los SRLV, todos ellos flanqueados por el regulador de la transcripción viral, la región LTR. En la patogénesis de las enfermedades lentivirales es esencial conocer las interacciones entre el virus y el hospedador que determinan el tropismo y el desarrollo de los síntomas. Los factores virales incluyen las proteínas de la envoltura, encargadas de unirse al receptor celular y la región LTR encargada de regular la actividad transcripcional. Los factores del hospedador son más diversos ya que pueden incluir todo el fondo genético de una raza o una población determinada capaz de restringir la replicación viral de manera efectiva. Uno de los pasos clave en el establecimiento de la infección es la superación de las barreras de la inmunidad innata, que reconocen directamente determinantes virales e inducen la eliminación del patógeno mediante factores de restricción como APOBEC3. La caracterización genética y virológica de las estirpes implicadas en los brotes epidemiológicos de artritis y encefalitis, puede aportar hallazgos esenciales en el conocimiento de la relación entre la secuencia genética de los aislados y la capacidad para establecer la infección en un tejido determinado. Por otro lado, componentes de la inmunidad innata del hospedador capaces de inhibir la replicación viral pueden ser también determinantes del tropismo. Por todo ello en esta tesis nos planteamos los siguientes estudios: a) Aislamiento y caracterización genética de estirpes implicadas en el brote artrítico. b) Aislamiento y caracterización genética de estirpes implicadas en el brote de encefalitis. c) Caracterización de la restricción de los SRLV por APOBEC3.Small ruminant lentiviruses (SRLV) include Visna Maedi virus (VMV) and caprine arthritis encephalitis virus (CAEV), infect sheep and goats and are widely distributed around the world causing a multisistemic disease of carpal joints, lungs, mammary gland and central nervous system. Losses derived from SRLV infection range from increased reposition rates in affected flocks to decreased animal production and low commercial value of the flock. SRLV‐induced disease is a slow process that affects mainly lungs and mammary gland in sheep from Spain, however new epidemiological outbreaks of arthritis and encephalitis in sheep have affected a high number of animals causing direct losses. Currently, 5 genotypes (A‐E) have been described showing a high genetic and biological variability. Genotypes A and B include classic VMV and CAEV strains respectively, and are widely distributed whereas genotypes C and E are geographically restricted. In the absence of treatments or protective vaccines, control measures are based on early detection of infected animals and culling. After infection, animals develop an antibody response unable to control viral replication, but indicative of viral infection. Thus, through the application of serological methods infection can be detected. The most currently used method is ELISA based on recombinant proteins and/or synthetic peptides. However, available tools are designed on the basis of a single strain that, together with the high variability present in SRLV hinder the detection of the whole SRLV antigenic spectrum. Lentiviral genomic organization is quite conserved among viruses within the genus. Thus, structural genes gag, pol and env encode capsid proteins, proteins that replicate and integrate viral genome and envelope proteins, respectively, plus accessory genes vpr, rev and vif in the case of SRLV, all of them flanked by the LTR as transcription regulator. Host‐virus interactions are essential in the development of lentiviral‐induced pathogenesis finally determining tropism and disease onset. Viral factors include Env proteins that mediate binding with cellular receptor, and the LTR region due to its control of transcriptional activity. Host factors may include all the genetic background belonging to a specific breed or animal population able to restrict viral replication efficiently. Transgression of innate immune barriers responsible for the direct recognition and elimination of the pathogen, such as APOBEC3, is one of the key steps to overcome in the establishment of infection. Genetic and virological characterization of the strains involved in the arthritic and encephalitic outbreaks may provide new insights in the association between genetics and the ability to establish a tissue targeted infection. On the other hand, innate immunity restriction factors, able to inhibit viral replication, are also determinants of viral tropism. Therefore in this Thesis the following studies were conducted: a) Isolation and genetic characterization of SRLV strains involved in the arthritic outbreak. b) Isolation and genetic characterization of SRLV involved in the encephalitis outbreak. c) Characterization of the SRLV restriction by APOBEC3.Comisión Interministerial de Ciencia y Tecnología (CICYT)Programa Oficial de Doctorado en Biotecnología (RD 1393/2007)Bioteknologiako Doktoretza Programa Ofiziala (ED 1393/2007